1. Field of the Invention
The invention relates to methods and compositions for modulating protein aggregation, useful for treatment of protein aggregation diseases and elucidation of cellular pathways involved in protein aggregation.
2. Description of the Related Art
Protein Aggregation Diseases
Human neurodegenerative disorders such as the tauopathies (including, e.g., Alzheimer's disease), Parkinson disease, amyotrophic lateral sclerosis, and polyglutamine expansion diseases (including, e.g., Huntington's disease and spinobulbar muscular atrophy) are all characterized by misfolding and aggregation of pathogenic proteins. These phenomena appear closely linked to pathology, although it has been unclear whether they represent a step in pathogenesis or even a protective mechanism. It is clear, however, that polyglutamine misfolding and aggregation occurs in a controlled fashion: it is noted primarily in affected neurons in patients, and is subject to regulation within the cell by various pathways (Chen et al., (2003) Cell 113, 457-468; Diamond et al., (2000). Proc Natl Acad Sci 97, 657-661; Emamian et al., (2003). Neuron 38, 375-387; Humbert et al., (2002). Dev Cell 2, 831-837; Meriin et al., (2001). J Cell Biol 153, 851-864; Wyttenbach et al. (2000). Proc Natl Acad Sci 97, 2898-2903).
Polyglutamine expansion diseases derive from CAG-codon expansion in certain genes. This enlarges a tract of glutamines in the target protein that produces neurodegeneration when it exceeds a critical threshold. Spinobulbar muscular atrophy (SBMA) is a progressive motor neuron disease caused by polyglutamine expansion in the N-terminus of the androgen receptor (AR) (Kennedy et al. (1968). Neurology 18, 671-680; La Spada et al., (1991). Nature 352, 77-79). Huntington's disease (HD) is a progressive neuropsychiatric degenerative condition with an associated movement disorder, and derives from a similar expansion in a protein of unknown function, huntingtin (htt) (The Huntington's Disease Collaborative Research Group (1993)).
Currently there are no effective therapies for HD, SBMA, or any neurodegenerative disease. Thus, methods to discover therapeutic targets and/or small molecules and other compounds, e.g., peptides, nucleic acids, and the like, that modulate pathogenesis are of crucial importance.
Cellular toxicity in most protein aggregation diseases correlates with nuclear accumulation and inclusion formation of mutant protein, and, in certain cases, may derive from “toxic fragments” produced through proteolysis (DiFiglia et al., (1997). Science 277, 1990-1993; Ellerby et al., (1999). J Neurochem 72, 185-195; Hodgson et al., (1999). Neuron 23, 181-192; Li et al., (1998). Ann Neurol 44, 249-254; Merry et al., (1998). Hum Mol Genet 7, 693-701). Several lines of evidence suggest that cells form inclusions as a physiological response to pathogenic proteins. Neurons of the central nervous system form predominantly nuclear inclusions, especially in affected regions, whereas somatic tissues generally do not (DiFiglia et al., (1997). Science 277, 1990-1993; Li et al., (1998). Ann Neurol 44, 249-254; Paulson et al., (1997). Neuron 19, 333-344). Activation of stress pathways via heat shock or JNK activation increases polyglutamine protein aggregation (Cowan et al., (2003). Hum Mol Genet 12, 1377-1391; Meriin et al., (2001). J Cell Biol 153, 851-864; Wyttenbach et al. (2000). Proc Natl Acad Sci 97, 2898-2903). In response to activation of Akt kinase, one group has reported decreased htt toxicity and aggregation in cell models (Humbert et al., (2002). Dev Cell 2, 831-837), while others report increased ataxin-1 toxicity and aggregation (Chen et al., (2003). Cell 113, 457-468). Several reports indicate that cytoplasmic inclusion formation within cells is a microtubule-dependent process (Garcia-Mata et al., (1999). J Cell Biol 146, 1239-1254; Muchowski et al., (2002). Proc Natl Acad Sci 99, 727-732; Taylor et al., (2003). Hum Mol Genet 12, 749-757). It has also been demonstrated that over-expressed wild-type glucocorticoid receptor (GR) diminishes polyglutamine protein aggregation after activation by hormone agonist, whereas a mutant form (GRΔ) increases nuclear inclusion formation and polyglutamine-dependent toxicity (Diamond et al., (2000). Proc Natl Acad Sci 97, 657-661; Welch and Diamond, (2001). Hum Mol Genet 10, 3063-3074). Thus, polyglutamine protein conversion from a soluble to an aggregated form appears to be a highly regulated process within the cellular milieu, and is not driven simply by a propensity for self-association.
Current Assays
The cellular pathways and molecular mechanisms that regulate protein misfolding and aggregation in polyglutamine and other neurodegenerative diseases remain largely unknown. It would be useful to elucidate these pathways and mechanisms, so that they can be targeted to prevent disease progression.
A variety of high-throughput approaches have been applied to identify direct inhibitors of polyglutamine aggregation in vitro (Berthelier et al., (2001). Anal Biochem 295, 227-236; Georgalis et al., (1998). Proc Natl Acad Sci 95, 6118-6121; Wanker et al., (1999). Methods in Enzymology 309, 375-386). In these cases, formation of insoluble aggregates of purified peptides serves as a readout. However, these methods cannot uncover underlying mechanisms that regulate intracellular protein misfolding and aggregation.
Yeast assays to study protein aggregation have been developed that express polyglutamine domains (Krobitsch and Lindquist. (2000). PNAS 97(4): 1589-94.) However, the resulting aggregates are non-toxic, raising questions about the applicability of the yeast cells as a model for human disease, e.g., polyglutamine expansion diseases.
In cultured cells, two basic approaches have been used to detect aggregation: identification of macro-aggregates by microscopy, by detergent insolubility, or by failure to pass through a membrane of fixed pore size (“filter trap”). However, such methods are highly labor-intensive, can be prone to experimenter bias, and are not particularly quantitative. Recently Kim et al. used fluorescence resonance energy transfer (FRET) combined with single cell imaging to study the constituents of large macro-molecular aggregates in cultured cells (Kim et al., (2002). Nat Cell Biol 4, 826-831). This method, while providing relatively precise spatial resolution, is quite time- and labor-intensive, and does not quantify the degree of polyglutamine protein aggregation within a population of cells. Moreover, by focusing only on large inclusions, this and other microscopy-based approaches ignore small oligomeric micro-aggregates that can play an important role in pathogenesis (Taylor et al., (2003). Hum Mol Genet 12, 749-757). Currently there are no facile, high-throughput methods with which to identify cellular pathways and molecular mechanisms that regulate protein misfolding and aggregation in polyglutamine expansion diseases and other neurodegenerative diseases in animal cells, e.g., in situ.